Monday, July 23, 2007

The Story of DNA (Part 2)

We've discovered the secret of life.

.... Francis CrickWhere Jim and Francis Discover the Secret of Life

Following the disaster of their first attempt at a DNA structure, Francis Crick went back to studying proteins [The Story of DNA (Part 1)]. He and William Cochran worked out the theory of the X-ray diffraction pattern of helices. Crick became the leading expert on the interpretation of patterns due to helices and he was able to predict what kind of pattern a particular helical pattern would show. This study paid huge dividends later on. Crick also worked out the coiled coil arrangement of polypeptide chains.

Watson dabbled in a number of projects over the next year. Most importantly, he had Crick teach him diffraction theory and he applied it to the structure of tobacco mosaic virus showing that it was a helix. Watson too became extremely adept at recognizing helices from their X-ray diffraction pattern.

Franklin made one important discovery. She showed that there were two distinct forms of DNA and that the original Astbury pictures were composites of the two forms. She called them A and B and those are the same names that we give them today. The B form is the naturally occurring form and the DNA has to maintained at high humidity in order to persist in this form The A form is somewhat dehydrated, if the fibers dry out, the structure converts to the A form.

On April 10, 1952 Rosalind Franklin took a picture of the A form of DNA. This picture was complex but it had some significant new features. Franklin came to rely heavily on the images of dehydrated DNA (A-DNA). Over the spring and summer she convinced herself that DNA was not helical. In fact, on July 18th, 1952 she and Gosling announced “the death of the helix” by sending out small cards with black borders [Rosalind Franklin Anounces the Death of the Helix]..

Recall that Raymond Gosling was the former graduate student of Wilkins, now assigned to Franklin. Franklin was rapidly making herself a real pain in the you-know-what but everybody loved Raymond.

On May 2 and May 6, 1952, while immersed in the analysis of the A form of DNA, Franklin took two beautiful pictures of B-DNA (right). The photos screamed helix. A cursory glance by someone familiar with helical diffraction patterns showed that the bases were 0.34 nm apart; that there were ten nucleotides per turn; that each turn was 3.4 nm; that the phosphate groups were on the outside; that the diameter of the helix was 2 nm; and that there were most likely two polynucleotide strands. Rosalind Franklin did not recognize these features and she put the photos aside.

After announcing the death of the helix, Franklin set out to secure herself a new job at another institute. It is clear that she did not think that the structure of DNA was very important. She soon received an offer to move to Birkbeck College, London but delayed until the following spring. Meanwhile, she set herself the task of working out the structure directly from the X-ray diffraction patterns. She refused to engage in speculation or model building and preferred to try and let the data lead her directly to the correct structure. By January of 1953 she knew that she was not going to solve DNA and she prepared to abandon the problem and publish the data she had obtained.

With hindsight, it’s clear that Franklin needed a trusted collaborator in order to make progress on this difficult problem. While working in Wilkins group she found herself isolated because she and Wilkins did not get along. (Both were to blame.) Later on in her career she collaborated effectively with Aaron Klug and Francis Crick.

Franklin also proved that the unit cell of the DNA fibers was monoclinic, face centered. This was an absolutely crucial piece of information but one that Franklin failed to appreciate. As soon as Crick became aware of it, in January 1953, an important part of the structure became apparent (see below).

What were Watson and Crick up to in the summer of 1952 when Rosalind Franklin was announcing the death of the helix? Well, for one thing they were not ignoring DNA in spite of Bragg’s warning.

Crick had begun to consider the possibility that the bases might be on the inside of the helix. He asked his friend, John Griffith, to do some calculations to see whether the bases could interact with one another. Griffith replied that A and T are compatible and so are G and C. This has nothing to do with hydrogen bonds—that would come later—but it did confirm one idea in Crick’s mind. Both he and Watson were familiar with the idea of complementarity. Crick thought of it as a way of explaining how DNA was replicated since one part of DNA could give rise to its complement while the other could make the second part. Complementarity was often discussed among the phage group since Delbruck and Linus Pauling had published a paper on it just before the war.

They impressed me by their extreme ignorance … I never met two men who knew so little—and aspired to so much.

..... Erwin ChargaffErwin Chargaff visited Cambridge in May 1952. Chargaff met with Watson and Crick and explained his work on the base composition of DNA. The results were new to Crick but known to Watson. Crick immediately saw that A=T and G=C and that fitted in with his ideas about complementarity.

Chargaff was not impressed. He said later on that, "They impressed me by their extreme ignorance ... I never met two men who knew so little—and aspired to so much." Later on after the structure of DNA had been published Chargaff said, "That in our day such pygmies throw such giant shadows only shows how late in the day it has become."

After a round of conferences in the summer, life began to settle down again at the Cavendish labs in Cambridge. Watson and Crick were joined by two new members of the lab. Peter Pauling, the son of Linus Pauling, had become a graduate student and brought news from his father that Pauling senior was thinking about DNA. The other new member of the group was Jerry Donohue. He was a former graduate student of Pauling's who was joining Bragg's group as a post-doc.

On Wednesday, January 28th, 1953 a copy of the Pauling and Cory manuscript on the structure of DNA reached Watson and Crick. The structure was wrong. In fact, it was similar to the Watson and Crick model that Rosalind Franklin had destroyed fourteen months earlier. Watson and Crick were elated and they determined to try again in spite of the ban imposed by Bragg. (The ban was soon to be lifted.)

On Friday, January 30, 1953 Watson was in London and he stopped by to see Franklin in her lab (left). Watson showed her a copy of the Pauling and Cory manuscript and she too saw that it was wrong. Watson began lecturing Franklin about helices—remember that Franklin was, at this time, concentrating on the A form of DNA and had all but ruled out that it was a helix. However, she was beginning to have some doubts about her hasty announcement of the death of the helix [Rosalind Franklin Announces the Death of the Helix]. She resented Watson's lecture and advanced toward Watson with a view to dismissing him. Watson beat a hasty retreat. (Jim Watson is well over six feet tall and Rosalind Franklin is very much shorter.)

At that moment Wilkins came by and he and Watson walked off comforting one another in the knowledge that Franklin was impossible. Wilkins told Watson about the excellent pictures of B DNA that Franklin had taken eight months ago (May 1952). He showed Watson one of the pictures (see above)). Watson left London with the knowledge that the B form of DNA was unmistakably helical, that the diameter was 2Å (2 nm), that there were 10 bases per turn, and that one turn was 34Å (3.4 nm). Some of this he got from the photo and some from measurements that Wilkins himself had made.

With this information, Watson started to build models. He began with the backbones inside but soon realized that it was impossible. Crick urged him to try to put the bases inside. Franklin had already concluded from her data that the phosphates were on the outside but it's not clear that Watson and Crick knew this.

Now comes a crucial bit of information. Rosalind Franklin had written a summary of her results for an institute report in December. Perutz gave Crick a copy. In that report Crick read for the first time that the crystalline form of DNA was based on a face-centered monoclinic unit cell. Why is this important?

It's important because such a unit cell has a two-fold axis of symmetry. That means that the molecule looks the same whether it is right way up or upside down. This has important implications for the two strands of DNA. To see this, think about two pencils side-by-side with the points down and the erasers on top. If you turn the two pencils upside down they look very different. Now the tips are pointing upward. However, if you line up the two pencils side-by-side with the tip of one pointing up and the tip of the other pointing down, when you flip the pair upside down they look the same. It means the two strands of DNA must be anti-parallel [The Chemical Structure of Double-Stranded DNA].

The space group of Franklin's DNA just happened to be the same space group as that of hemoglobin, the molecule that Crick was working on as the subject of his Ph.D. thesis. Crick recognized immediately what this meant.

Watson worked out another argument that convinced them that there had to be two chains in the unit cell and not three. It had to do with the density and water content and we won't go into it here. Suffice to say that in the last days of February they knew that the backbones were on the outside, that there were two chains, and that the chains ran in opposite directions.

On Friday, February 20th Watson presented some ideas about base pairs to his colleagues. He had come up with a scheme involving like pairs (A/A, G/G etc.). Jerry Donohue instantly recognized a problem. Watson was using the standard textbook structures of the bases, the imino and lactim tautomers. Donohue knew that the predominant forms in living cells were the other tautomers, the amino and lactam conformations [Tautomers of Adenine, Cytosine, Guanine, and Thymine]. This was the final important clue. Like pairing with like was not an option; besides, it didn't conform to Chargaff's rules.

The next week Watson made some cardboard cutouts of the bases and began to try and fit them together into the middle of the backbones running in opposite directions. Crick urged him to think about complementarity—recall that the previous summer Crick had convinced himself that complementarity was the key to DNA replication. He had forgotten about the A=T and G=C data from Chargaff.

On Saturday, February 28, Watson was playing with his cardboard cutouts when he discovered that you could fit A/T and G/C base pairs into the model. Crick immediately confirmed that this was an elegant solution. They then realized that it explained the Chargaff ratios.

It took them about a week to build a detailed model. Many experts were called to give their opinion and all pronounced it sound. Wilkins, Gosling, and Franklin came up to Cambridge to see the model and agreed that it must be right. Raymond Gosling is an admirer of Wilkins and in reviewing Wilkins' autobiography in Nature (Gosling, 2003) Gosling writes,

Wilkins eloquently describes his feelings at seeing the double-helix structure for the first time: "It seemed that non-living atoms and chemical bonds had come together to form life itself. I was rather stunned by it all." This sums up beautifully how Franklin and I felt. It was so elegant an explanation of all of the complex properties required of DNA, and contained so many elements familiar from our own work using X-ray diffraction. At the time I did not know that Wilkins was offered co-authorship by Watson and Crick, but refused. It would certainly have been appropriate, and seems to be something that he later came to regret.

The paper was written up and sent off to Nature on April 2. It was published on April 25, 1953 along with papers by Franklin and Wilkins.

Before publication, Linus Pauling visited Cambridge and confirmed that the Watson/Crick model was correct and his model was wrong. The first announcement of the discovery was made by Bragg at a conference in Brussels in early April.

Laurence A. Moran

Larry Moran is a Professor in the Department of Biochemistry at the University of Toronto. You can contact him by looking up his email address on the University of Toronto website.

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Quotations

The old argument of design in nature, as given by Paley, which formerly seemed to me to be so conclusive, fails, now that the law of natural selection has been discovered. We can no longer argue that, for instance, the beautiful hinge of a bivalve shell must have been made by an intelligent being, like the hinge of a door by man. There seems to be no more design in the variability of organic beings and in the action of natural selection, than in the course which the wind blows.Charles Darwin (c1880)Although I am fully convinced of the truth of the views given in this volume, I by no means expect to convince experienced naturalists whose minds are stocked with a multitude of facts all viewed, during a long course of years, from a point of view directly opposite to mine. It is so easy to hide our ignorance under such expressions as "plan of creation," "unity of design," etc., and to think that we give an explanation when we only restate a fact. Any one whose disposition leads him to attach more weight to unexplained difficulties than to the explanation of a certain number of facts will certainly reject the theory.

Charles Darwin (1859)Science reveals where religion conceals. Where religion purports to explain, it actually resorts to tautology. To assert that "God did it" is no more than an admission of ignorance dressed deceitfully as an explanation...

Quotations

The world is not inhabited exclusively by fools, and when a subject arouses intense interest, as this one has, something other than semantics is usually at stake.
Stephen Jay Gould (1982)
I have championed contingency, and will continue to do so, because its large realm and legitimate claims have been so poorly attended by evolutionary scientists who cannot discern the beat of this different drummer while their brains and ears remain tuned to only the sounds of general theory.
Stephen Jay Gould (2002) p.1339
The essence of Darwinism lies in its claim that natural selection creates the fit. Variation is ubiquitous and random in direction. It supplies raw material only. Natural selection directs the course of evolutionary change.
Stephen Jay Gould (1977)
Rudyard Kipling asked how the leopard got its spots, the rhino its wrinkled skin. He called his answers "just-so stories." When evolutionists try to explain form and behavior, they also tell just-so stories—and the agent is natural selection. Virtuosity in invention replaces testability as the criterion for acceptance.
Stephen Jay Gould (1980)
Since 'change of gene frequencies in populations' is the 'official' definition of evolution, randomness has transgressed Darwin's border and asserted itself as an agent of evolutionary change.
Stephen Jay Gould (1983) p.335
The first commandment for all versions of NOMA might be summarized by stating: "Thou shalt not mix the magisteria by claiming that God directly ordains important events in the history of nature by special interference knowable only through revelation and not accessible to science." In common parlance, we refer to such special interference as "miracle"—operationally defined as a unique and temporary suspension of natural law to reorder the facts of nature by divine fiat.
Stephen Jay Gould (1999) p.84

Quotations

My own view is that conclusions about the evolution of human behavior should be based on research at least as rigorous as that used in studying nonhuman animals. And if you read the animal behavior journals, you'll see that this requirement sets the bar pretty high, so that many assertions about evolutionary psychology sink without a trace.

Jerry Coyne
Why Evolution Is TrueI once made the remark that two things disappeared in 1990: one was communism, the other was biochemistry and that only one of them should be allowed to come back.

Sydney Brenner
TIBS Dec. 2000
It is naïve to think that if a species' environment changes the species must adapt or else become extinct.... Just as a changed environment need not set in motion selection for new adaptations, new adaptations may evolve in an unchanging environment if new mutations arise that are superior to any pre-existing variations

Douglas Futuyma
One of the most frightening things in the Western world, and in this country in particular, is the number of people who believe in things that are scientifically false. If someone tells me that the earth is less than 10,000 years old, in my opinion he should see a psychiatrist.

Francis Crick
There will be no difficulty in computers being adapted to biology. There will be luddites. But they will be buried.

Sydney Brenner
An atheist before Darwin could have said, following Hume: 'I have no explanation for complex biological design. All I know is that God isn't a good explanation, so we must wait and hope that somebody comes up with a better one.' I can't help feeling that such a position, though logically sound, would have left one feeling pretty unsatisfied, and that although atheism might have been logically tenable before Darwin, Darwin made it possible to be an intellectually fulfilled atheist

Richard Dawkins
Another curious aspect of the theory of evolution is that everybody thinks he understand it. I mean philosophers, social scientists, and so on. While in fact very few people understand it, actually as it stands, even as it stood when Darwin expressed it, and even less as we now may be able to understand it in biology.

Jacques Monod
The false view of evolution as a process of global optimizing has been applied literally by engineers who, taken in by a mistaken metaphor, have attempted to find globally optimal solutions to design problems by writing programs that model evolution by natural selection.